杜氏盐藻完整叶绿体的分离和叶绿体转化体系的构建
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摘要
随着转基因研究的不断深入，人们将越来越多的研究兴趣集中在生长迅速，培养简单的单细胞绿藻上。利用单细胞绿藻进行遗传转化有其优越性，与传统的微生物发酵、动物细胞和转基因动物等转化系统相比，它不需要昂贵的设备和严格的培养条件，具有光合自养、培养成本低，易于转化等优点，是一类比较廉价和安全的生物反应器。
目前，对单细胞绿藻的转化工作主要集中在两大方向：细胞核转化和叶绿体转化。经过数十年的发展，植物的核转化技术日臻成熟并得到了广泛的应用，但是核基因组的遗传转化仍存在一系列至今尚未解决的内在问题：如由于核基因组大、背景复杂、易出现基因失活、基因沉默、位置效应等现象；所表达的原核基因必须经过修饰改造，环境安全难以保证等。
而在叶绿体转化系统中却可以克服这些困难。与核转化相比，叶绿体基因组小，遗传操作简单，外源基因是通过同源重组机制定点整合进叶绿体基因组。定点整合有利于人为控制外源基因的插入位点，可以将目的基因定位在适于表达的位点，能较好地解决“顺式失活”、“位置效应”等类的基因沉默问题。由于叶绿体中DNA分子有多个拷贝，同时叶绿体对表达产物的积累有较强的承受能力，保障了外源基因在叶绿体中的高效表达。另外，由于叶绿体基因组的原核性质，对来自原核生物的外源基因无需改造就可以在叶绿体内高效表达，而且可以将多个外源基因采取“多顺反子”的原核表达形式同时引入，并由共同的启动子控制，既方便操作又可避免由于存在多个相同启动子所带来的“共沉默”。这是核基因转化无法做到的。
杜氏盐藻(以下简称盐藻)是一种低等的单细胞真核绿藻，属绿藻门绿藻纲团藻目，进化树分析显示其与具有细胞壁的莱茵衣藻十分相似，没有细胞壁，长约6-15μm，呈椭圆形或梨形，能依靠其双鞭毛在水中游动。光镜下可明显看到其内含一个大型的杯状叶绿体，约占细胞总体积的50％。可在0.05M-5.5M的盐水中生长，最佳生长繁殖盐度为2M-3M，目前已知最耐盐的真核生物。由于盐藻可在高渗盐溶液中生长，这是许多其它生物难以生存的环境，故其大规模培养不需昂贵的发酵罐或其他培养装置，可以直接采用开放式培养，大大降低生产成本，由于没有细胞壁可很容易的用基因枪，电击法等一系列转化方法进行遗传转化，这些独特使得杜氏盐藻可被开发为一种生产药用蛋白的良好宿主。
基于叶绿体转化的优越性以及盐藻的独特性，本实验意欲在盐藻中实现稳定高效的叶绿体转化。在我们以前的实验中，初步的盐藻叶绿体转化系统已经构建成功，但是由于用于重组的同源片段长度不足使得得到的转化系统不太稳定。本实验首先分离大量完整的叶绿体，进而抽提高纯度叶绿体DNA(cpDNA)，利用四个平端酶随机切割cpDNA并连上特异设计的接头(Linker)，构建四个叶绿体基因组步行文库，通过巢式PCR扩增到足够长度的同源片段，进而构建叶绿体转化载体。
实验方法
1．盐藻完整叶绿体的分离及cpDNA的提取
在进行盐藻叶绿体的分离时，从某种程度上讲对其大量高效的培养显得至关重要。合适的培养条件一方面可以使细胞快速增殖，另一方面则可以保持整个生长过程中叶绿体保持良好的形态。我们在比较了不同的盐藻生长的培养基的基础上，对UTEX液体培养液进行了部分改良，如将NaCl的浓度调至1M。
取对数生长后期的杜氏盐藻细胞进行高压破碎，利用差速离心和蔗糖密度梯度离心得到完整的叶绿体。采用优化的SDS-proteinase K-酚／氯仿／异戊醇方案抽提得到高纯度的cpDNA。
2．盐藻chlN基因未知侧翼区的扩增
chlN被选择用于我们进行叶绿体转化的同源片段。它和chlL、chlB参与编码光非依赖性的原叶绿素酸醋还原酶(DPOR)，任何一个基因的破坏都阻断黑暗条件下叶绿素的合成。chlL基因插入失活后，盐藻还可通过光依赖性的原叶绿素酸醋还原酶系统(LPOR)完成叶绿素的合成，而不影响转化藻的正常生存。
2．1盐藻叶绿体基因组步行文库的构建
将抽提得到的高纯度cpDNA分别用DraⅠ、EcoRⅤ、PvuⅡ和StuⅠ四个平端限制酶切，之后和特异的接头连接制成叶绿体基因组步行文库，作为巢式PCR扩增的模板。
2．2 chlN基因未知上游侧翼的扩增
通过在chlN基因上游设计特异引物，在制成的叶绿体基因组步行文库中进行chlN基因未知上游的扩增。
2．3 chlN基因未知下游侧翼的扩增
同样，通过在chlN基因下游设计特异引物，在叶绿体基因组步行文库中进行chlN基因未知下游的扩增。
3．盐藻双交换叶绿体转化载体的构建及转化结果的初步鉴定
在新增同源片段的基础上，对已有的叶绿体转化载体进行改造，构建成盐藻叶绿体转化载体pMDko-bar以用于叶绿体转化。用电击转化法转化野生型盐藻细胞。
实验结果
1．盐藻完整叶绿体的分离及高纯度cpDNA的制备
蔗糖密度梯度离心结果表明分离到了大量的叶绿体，相差显微镜及电镜观察证实了所得叶绿体的完整性。1％琼脂糖凝胶电泳检测cpDNA，并用紫外分光光度仪检测cpDNA的浓度和纯度，结果显示，抽提到的cpDNA浓度约2.20μg／ml，A260／A280接近1.8，说明其含量及纯度都达到了比较理想的状态。
2．盐藻chlN基因未知侧翼区的扩增
2．1 chlN基因未知上游侧翼的扩增
经过两轮的扩增得到一条3000 bp的片段，测序结果显示其3’端与以前实验中用简并引物扩增得到的1269 bp的chlN的5’端完全吻合。
2．1 chlN基因未知下游侧翼的扩增
两轮的扩增得到一条650 bp的片段，测序结果显示其5’端与已知的1269 bp的chlN的3’端完全吻合。
3．盐藻双交换叶绿体转化载体的构建及转化的初步鉴定
酶切鉴定双交换叶绿体转化载体pMDko-bar插入片段大小、方向均正确。转化过的藻株在筛选压力下，于一周后肉眼观察到转化藻株与阴性对照藻株(未加质粒，电击转化后加入PPT)生长状态存在明显差异。而与阳性对照(未加质粒，电击转化后不加PPT)的生长状态并无很大差别。则可以推知已将目的基因成功转入盐藻叶绿体。
结论
1．在分离到了大量完整的盐藻叶绿体基础上，通过对传统方案的优化得到足量的高纯度cpDNA。
2．从构建的盐藻叶绿体基因组步行文库中成功扩增得到用于叶绿体遗传转化的同源片段，并构建了叶绿体转化载体。
3．转化结果显示目的基因已转入盐藻叶绿体基因组。
Recently, the research focusing on genetic transformation of algae has been carried out intensively, and some of them have been developed as new type bioreactors for the production of pharmaceutical proteins or vaccines. Compared to traditional transformational systems including microbial fermentation, transgenic animals and plants, transgenic algae have some particular advantages, for example, they are autotrophy and do not require expensive media or strict culture conditions. Because of these unique characteristics, the algae are developed to low-cost and safe bioreactors.
Nowadays, studies for the genetic transformation of unicellular eukaryotic algae mainly focus on two approaches: nucleus and chloroplast transformation. Though the nucleic transformation in plants has been developed and used widely, some intrinsic problems in genetic information have not been solved. The nucleic genome is so big and complicated that the integration sites and copies of foreign genes can not be controlled accurately, leading to the inefficient expression of the foreign genes because of gene silencing.
The chloroplast transformation may solve some of the problems mentioned above. Compared to nucleic genome, the chloroplast genome is smaller and easier to be operated genetically, and the foreign gene is site-direct integrated into the chloroplast genome through homologous recombination. Besides, there are many copies of chloroplast DNA(cpDNA) in single chloroplast, and the chloroplast has a strong tolerance to accumulation of the products expressed by the introduced gene, which will ensure the high efficient expression of the foreign gene. Meanwhile, the prokaryotic genes are efficient expressed in the chloroplast without any modification because of prokaryotic property of the chloroplast transformation, but not in nucleic transformation.
Dunaliella salina (D. salina) belongs to Chlorophyta, Chlorophycease, Volvocales. Its shape and structure are very similar to Chlamydomonas reinhardtii (C. reinhardtii) except for lacking of cell wall. It has a large cup-shaped chloroplast about 50% of the cell volume. The cells are able to grow in extreme environments such as in a variety of sodium chloride concentration ranging from 0.05M to 5M, where other organisms hardly survive, so large-scale cultures of D. salina do not need expensive equipments, suggesting that D.salina is a favorable host for producing pharmaceutical proteins.
In this study, after a chloroplast genome walking library was constructed, target homologous DNA fragments were obtained by using nested PCR, and finally a vector for chloroplast transformation was successfully constructed.
Methods:
1. Isolation of the intact chloroplast from D.salina and extraction of cpDNA
The cells grown at -27℃in UTEX medium containing 1M NaCl, and a light/dark regime of 12/12h was selected. Before harvesting, cultures were examined microscopically for contamination. Subsequently, exponentially growing cultures, about 7 days after inoculation, were harvested and broken through cell bomb. The intact chloroplasts were obtained by differential centrifugation and sucrose density gradient centrifugation. A modified scheme of SDS-proteinase K-phenol/chloroform/isoamyl alcohol was employed to extract cpDNA.
2. Amplification of uncharacterized flanking regions of the chlN gene
chlN together with chlL and chlB are responsible for the biosynthesis of light-independent protochlorophyllide reductase (LIPOR) of the chloroplasts in D. salina cells. D. salina cells can also perform chlorophyll formation through light-dependent protochlorophyllide reductase (LDPOR) even without chlN gene. So the loci of the chlN genes were selected as the homologous recombination regions.
2.1 Construction of chloroplast genome walking libraries
Four chloroplast genome walking libraries DL, EL, PL and SL of D. salina were constructed after the cpDNA digested by Dra I, EcoR V, Pvu II and Stu I was ligated to designed adaptors.
2.2 Amplication of uncharacterized flanking regions of the chlN genes
Primers used in the amplification were designed according to the upstream and downstream sequences of the chlN gene, and the chloroplast genome walking libraries were served as the templates. The nested PCR was selected for amplification of uncharacterized flanking regions of the chlN genes.
3. Construction of vector for the chloroplast transformation and identification for result of transformation
A new vector named as pMDko-bar for chloroplast transformation had been constructed after the homologous recombination fragments were obtained, and then the pMDko-bar was introduced into wild type cells of D. salina by electroporation.
Result:
1. Isolation of the intact chloroplast from D.salina and extraction of cpDNA
Result of sucrose density gradient centrifugation revealed that a mass of chloroplasts were isolated. The integrity of the chloroplasts was confirmed with phase contrast microscope and electronic microscope. The results of examination for the concentration and purity of the cpDNA through an ultraviolet spectrophotometer and 1 % agarose gels were satisfactory.
2. Amplification of uncharacterized flanking regions of the chlN gene
The result of nested PCR indicated that 600 bp and 3000 bp of fragments were amplified from the upstream chlN, and 650 bp of fragment was amplified from the downstream chlN through genome walking technique. The sequencing results showed that the amplified fragments were correct.
3.Construction of vector for the chloroplast transformation and identification for result of transformation
The identification for vector pMDko-bar indicated that both the size and direction of the inserted fragment were correct. One week later, the transformed cells were able to grow under the select pressure of 0.2μg/ml PPT, while the negative control hardly survived. The above observation suggests that the target gene has been introduced into the chloroplast of D. salina cells.
Conclusion:
1. cpDNA with high purity has been extracted after intact chloroplasts wereisolated from D. salina cells.
2. A vector pMDko-bar for chloroplast transformation has been successfully constructed after homologous fragments were obtained.
3.identification results reveal that the target gene has been introduced into the chloroplasts of D. salina cells.
目前，对单细胞绿藻的转化工作主要集中在两大方向：细胞核转化和叶绿体转化。经过数十年的发展，植物的核转化技术日臻成熟并得到了广泛的应用，但是核基因组的遗传转化仍存在一系列至今尚未解决的内在问题：如由于核基因组大、背景复杂、易出现基因失活、基因沉默、位置效应等现象；所表达的原核基因必须经过修饰改造，环境安全难以保证等。
而在叶绿体转化系统中却可以克服这些困难。与核转化相比，叶绿体基因组小，遗传操作简单，外源基因是通过同源重组机制定点整合进叶绿体基因组。定点整合有利于人为控制外源基因的插入位点，可以将目的基因定位在适于表达的位点，能较好地解决“顺式失活”、“位置效应”等类的基因沉默问题。由于叶绿体中DNA分子有多个拷贝，同时叶绿体对表达产物的积累有较强的承受能力，保障了外源基因在叶绿体中的高效表达。另外，由于叶绿体基因组的原核性质，对来自原核生物的外源基因无需改造就可以在叶绿体内高效表达，而且可以将多个外源基因采取“多顺反子”的原核表达形式同时引入，并由共同的启动子控制，既方便操作又可避免由于存在多个相同启动子所带来的“共沉默”。这是核基因转化无法做到的。
杜氏盐藻(以下简称盐藻)是一种低等的单细胞真核绿藻，属绿藻门绿藻纲团藻目，进化树分析显示其与具有细胞壁的莱茵衣藻十分相似，没有细胞壁，长约6-15μm，呈椭圆形或梨形，能依靠其双鞭毛在水中游动。光镜下可明显看到其内含一个大型的杯状叶绿体，约占细胞总体积的50％。可在0.05M-5.5M的盐水中生长，最佳生长繁殖盐度为2M-3M，目前已知最耐盐的真核生物。由于盐藻可在高渗盐溶液中生长，这是许多其它生物难以生存的环境，故其大规模培养不需昂贵的发酵罐或其他培养装置，可以直接采用开放式培养，大大降低生产成本，由于没有细胞壁可很容易的用基因枪，电击法等一系列转化方法进行遗传转化，这些独特使得杜氏盐藻可被开发为一种生产药用蛋白的良好宿主。
基于叶绿体转化的优越性以及盐藻的独特性，本实验意欲在盐藻中实现稳定高效的叶绿体转化。在我们以前的实验中，初步的盐藻叶绿体转化系统已经构建成功，但是由于用于重组的同源片段长度不足使得得到的转化系统不太稳定。本实验首先分离大量完整的叶绿体，进而抽提高纯度叶绿体DNA(cpDNA)，利用四个平端酶随机切割cpDNA并连上特异设计的接头(Linker)，构建四个叶绿体基因组步行文库，通过巢式PCR扩增到足够长度的同源片段，进而构建叶绿体转化载体。
实验方法
1．盐藻完整叶绿体的分离及cpDNA的提取
在进行盐藻叶绿体的分离时，从某种程度上讲对其大量高效的培养显得至关重要。合适的培养条件一方面可以使细胞快速增殖，另一方面则可以保持整个生长过程中叶绿体保持良好的形态。我们在比较了不同的盐藻生长的培养基的基础上，对UTEX液体培养液进行了部分改良，如将NaCl的浓度调至1M。
取对数生长后期的杜氏盐藻细胞进行高压破碎，利用差速离心和蔗糖密度梯度离心得到完整的叶绿体。采用优化的SDS-proteinase K-酚／氯仿／异戊醇方案抽提得到高纯度的cpDNA。
2．盐藻chlN基因未知侧翼区的扩增
chlN被选择用于我们进行叶绿体转化的同源片段。它和chlL、chlB参与编码光非依赖性的原叶绿素酸醋还原酶(DPOR)，任何一个基因的破坏都阻断黑暗条件下叶绿素的合成。chlL基因插入失活后，盐藻还可通过光依赖性的原叶绿素酸醋还原酶系统(LPOR)完成叶绿素的合成，而不影响转化藻的正常生存。
2．1盐藻叶绿体基因组步行文库的构建
将抽提得到的高纯度cpDNA分别用DraⅠ、EcoRⅤ、PvuⅡ和StuⅠ四个平端限制酶切，之后和特异的接头连接制成叶绿体基因组步行文库，作为巢式PCR扩增的模板。
2．2 chlN基因未知上游侧翼的扩增
通过在chlN基因上游设计特异引物，在制成的叶绿体基因组步行文库中进行chlN基因未知上游的扩增。
2．3 chlN基因未知下游侧翼的扩增
同样，通过在chlN基因下游设计特异引物，在叶绿体基因组步行文库中进行chlN基因未知下游的扩增。
3．盐藻双交换叶绿体转化载体的构建及转化结果的初步鉴定
在新增同源片段的基础上，对已有的叶绿体转化载体进行改造，构建成盐藻叶绿体转化载体pMDko-bar以用于叶绿体转化。用电击转化法转化野生型盐藻细胞。
实验结果
1．盐藻完整叶绿体的分离及高纯度cpDNA的制备
蔗糖密度梯度离心结果表明分离到了大量的叶绿体，相差显微镜及电镜观察证实了所得叶绿体的完整性。1％琼脂糖凝胶电泳检测cpDNA，并用紫外分光光度仪检测cpDNA的浓度和纯度，结果显示，抽提到的cpDNA浓度约2.20μg／ml，A260／A280接近1.8，说明其含量及纯度都达到了比较理想的状态。
2．盐藻chlN基因未知侧翼区的扩增
2．1 chlN基因未知上游侧翼的扩增
经过两轮的扩增得到一条3000 bp的片段，测序结果显示其3’端与以前实验中用简并引物扩增得到的1269 bp的chlN的5’端完全吻合。
2．1 chlN基因未知下游侧翼的扩增
两轮的扩增得到一条650 bp的片段，测序结果显示其5’端与已知的1269 bp的chlN的3’端完全吻合。
3．盐藻双交换叶绿体转化载体的构建及转化的初步鉴定
酶切鉴定双交换叶绿体转化载体pMDko-bar插入片段大小、方向均正确。转化过的藻株在筛选压力下，于一周后肉眼观察到转化藻株与阴性对照藻株(未加质粒，电击转化后加入PPT)生长状态存在明显差异。而与阳性对照(未加质粒，电击转化后不加PPT)的生长状态并无很大差别。则可以推知已将目的基因成功转入盐藻叶绿体。
结论
1．在分离到了大量完整的盐藻叶绿体基础上，通过对传统方案的优化得到足量的高纯度cpDNA。
2．从构建的盐藻叶绿体基因组步行文库中成功扩增得到用于叶绿体遗传转化的同源片段，并构建了叶绿体转化载体。
3．转化结果显示目的基因已转入盐藻叶绿体基因组。
Recently, the research focusing on genetic transformation of algae has been carried out intensively, and some of them have been developed as new type bioreactors for the production of pharmaceutical proteins or vaccines. Compared to traditional transformational systems including microbial fermentation, transgenic animals and plants, transgenic algae have some particular advantages, for example, they are autotrophy and do not require expensive media or strict culture conditions. Because of these unique characteristics, the algae are developed to low-cost and safe bioreactors.
Nowadays, studies for the genetic transformation of unicellular eukaryotic algae mainly focus on two approaches: nucleus and chloroplast transformation. Though the nucleic transformation in plants has been developed and used widely, some intrinsic problems in genetic information have not been solved. The nucleic genome is so big and complicated that the integration sites and copies of foreign genes can not be controlled accurately, leading to the inefficient expression of the foreign genes because of gene silencing.
The chloroplast transformation may solve some of the problems mentioned above. Compared to nucleic genome, the chloroplast genome is smaller and easier to be operated genetically, and the foreign gene is site-direct integrated into the chloroplast genome through homologous recombination. Besides, there are many copies of chloroplast DNA(cpDNA) in single chloroplast, and the chloroplast has a strong tolerance to accumulation of the products expressed by the introduced gene, which will ensure the high efficient expression of the foreign gene. Meanwhile, the prokaryotic genes are efficient expressed in the chloroplast without any modification because of prokaryotic property of the chloroplast transformation, but not in nucleic transformation.
Dunaliella salina (D. salina) belongs to Chlorophyta, Chlorophycease, Volvocales. Its shape and structure are very similar to Chlamydomonas reinhardtii (C. reinhardtii) except for lacking of cell wall. It has a large cup-shaped chloroplast about 50% of the cell volume. The cells are able to grow in extreme environments such as in a variety of sodium chloride concentration ranging from 0.05M to 5M, where other organisms hardly survive, so large-scale cultures of D. salina do not need expensive equipments, suggesting that D.salina is a favorable host for producing pharmaceutical proteins.
In this study, after a chloroplast genome walking library was constructed, target homologous DNA fragments were obtained by using nested PCR, and finally a vector for chloroplast transformation was successfully constructed.
Methods:
1. Isolation of the intact chloroplast from D.salina and extraction of cpDNA
The cells grown at -27℃in UTEX medium containing 1M NaCl, and a light/dark regime of 12/12h was selected. Before harvesting, cultures were examined microscopically for contamination. Subsequently, exponentially growing cultures, about 7 days after inoculation, were harvested and broken through cell bomb. The intact chloroplasts were obtained by differential centrifugation and sucrose density gradient centrifugation. A modified scheme of SDS-proteinase K-phenol/chloroform/isoamyl alcohol was employed to extract cpDNA.
2. Amplification of uncharacterized flanking regions of the chlN gene
chlN together with chlL and chlB are responsible for the biosynthesis of light-independent protochlorophyllide reductase (LIPOR) of the chloroplasts in D. salina cells. D. salina cells can also perform chlorophyll formation through light-dependent protochlorophyllide reductase (LDPOR) even without chlN gene. So the loci of the chlN genes were selected as the homologous recombination regions.
2.1 Construction of chloroplast genome walking libraries
Four chloroplast genome walking libraries DL, EL, PL and SL of D. salina were constructed after the cpDNA digested by Dra I, EcoR V, Pvu II and Stu I was ligated to designed adaptors.
2.2 Amplication of uncharacterized flanking regions of the chlN genes
Primers used in the amplification were designed according to the upstream and downstream sequences of the chlN gene, and the chloroplast genome walking libraries were served as the templates. The nested PCR was selected for amplification of uncharacterized flanking regions of the chlN genes.
3. Construction of vector for the chloroplast transformation and identification for result of transformation
A new vector named as pMDko-bar for chloroplast transformation had been constructed after the homologous recombination fragments were obtained, and then the pMDko-bar was introduced into wild type cells of D. salina by electroporation.
Result:
1. Isolation of the intact chloroplast from D.salina and extraction of cpDNA
Result of sucrose density gradient centrifugation revealed that a mass of chloroplasts were isolated. The integrity of the chloroplasts was confirmed with phase contrast microscope and electronic microscope. The results of examination for the concentration and purity of the cpDNA through an ultraviolet spectrophotometer and 1 % agarose gels were satisfactory.
2. Amplification of uncharacterized flanking regions of the chlN gene
The result of nested PCR indicated that 600 bp and 3000 bp of fragments were amplified from the upstream chlN, and 650 bp of fragment was amplified from the downstream chlN through genome walking technique. The sequencing results showed that the amplified fragments were correct.
3.Construction of vector for the chloroplast transformation and identification for result of transformation
The identification for vector pMDko-bar indicated that both the size and direction of the inserted fragment were correct. One week later, the transformed cells were able to grow under the select pressure of 0.2μg/ml PPT, while the negative control hardly survived. The above observation suggests that the target gene has been introduced into the chloroplast of D. salina cells.
Conclusion:
1. cpDNA with high purity has been extracted after intact chloroplasts wereisolated from D. salina cells.
2. A vector pMDko-bar for chloroplast transformation has been successfully constructed after homologous fragments were obtained.
3.identification results reveal that the target gene has been introduced into the chloroplasts of D. salina cells.
引文
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21．卢太白，曾玲，李坤，等。绿色蔬菜叶绿体DNA的提取和纯化。西北农业学报，2006，15(1)：186-188
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32．王关林，方宏。．植物基因工程，第二版，北京，科学出版社，2002年：330-336
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19．华栋，耿德贵。盐度对盐生杜氏藻叶绿体超微结构的影响。徐州师范大学学报(自然科学版)，1999，17(4)55
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21．卢太白，曾玲，李坤，等。绿色蔬菜叶绿体DNA的提取和纯化。西北农业学报，2006，15(1)：186-188
22．J 萨姆布鲁克，D W 拉塞尔．分子克隆实验指南．科学出版社，2002年8月第三版
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29. Staub TM, Maliga P Long region of homologous are incorporated into the tobacco plastid genome by transformation. Plant Cell, 1992, 4: 39-45.
30. Maliga P. Towards plastid transformation in flowering plants. TIBTECH, 1993, 11:101-107
31. Geng DG(耿德贵),Wang YQ(王义琴),Li WB(李文彬) et al. Transient expression of GUS Gene in Dunaliella Salina. High Technology Letters(高技术通讯), 2002,12(2):35-39
32．王关林，方宏。．植物基因工程，第二版，北京，科学出版社，2002年：330-336
1. Apt, K. E., Behrens, P.W. Commercial developments in microalgal biotechnology. J. Phycol. 1999, 35:215-26
2. Ben-Amotz A, Shaish A, Avron M. The biotechnology of cultivating Dunaliella for production of β-Carotene rich algae. Bioresource Technol, 1991,38:233-235
3. Richmond A. Handbook of microalgal culture:Biotechnology and applied phycology. USA:Blackwell Publishing Company,2003,359-360
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